Nuclear Transplantation in the Bovine Embryo: Assessment of Donor Nuclei and Recipient Oocyt14

TLDR

Blastomeres from 2‑ to 32‑cell bovine embryos were micromanipulated and electrofused into enucleated oocytes matured in vivo or in vitro, and the resulting nuclear‑transfer embryos were cultured in vitro or in vivo before being transferred into heifers. Fusion efficiency was higher with larger donor karyoplasts.

Abstract

Blastomeres from 2- to 32-cell bovine embryos were transferred to enucleated oocytes matured either in vivo or in vitro by micromanipulation and electrofusion. The percentage of donor cells fusing with the recipient oocytes was dependent on relative cell size or stage of development. Therefore, when smaller donor karyoplasts (17- to 32-cell vs. 2- to 8-cell) were transferred, the rate of fusion was significantly less (p < 0.01). After fusion, nuclear transfer embryos were cultured either in vitro or in vivo (in a ligated ovine oviduct). Nuclear transfer embryos cultured in vitro developed to the 4- to 6-cell stage after 72 h (4-cell, 71%; 8-cell, 33%, 16-cell, 33%; p < 0.30), whereas nuclear transfer embryos cultured in vivo developed to the morula or blastocyst stage (2- to 8-cell, 11.7%; 9- to 16-cell, 16.0%; 17- to 32-cell, 8.3%; p > 0.30) after 4 or 5 days. Freshly ovulated oocytes (collected 36 h after the onset of estrus), when used as recipients, resulted in morula/blastocyst-stage embryos more often than in vitro-matured oocytes or in vivo-matured oocytes collected 48 h after the onset of estrus (20% vs. 7.8% and 6.7%, respectively; p < 0.02). After in vivo culture, nuclear transfer embryos were mounted and fixed or transferred nonsurgically to the uteri of 6- to 8-day postestrus heifers. Seven pregnancies resulted from the transfer of 19 embryos into 13 heifers; 2 heifers completed pregnancy with the birth of live calves. These results suggest that the nuclei of early cleavage-stage bovine embryos can be reprogrammed to behave as nuclei of pronuclear stage embryos. Therefore, with increased efficiencies and in combination with serial nuclear transfers, it may be possible to produce multiple copies of a single embryo.